Natural mechanisms for generating antibody diversification exploit the process of somatic hypermutation (SHM) to trigger the evolution of immunoglobulin variable regions, thereby rapidly generating the secondary antibody repertoire associated with the humoral response. In vivo, SHM represents a highly efficient process, which is capable of rapidly exploring productive folding structures and evolving high affinity antibodies in a manner that represents the natural process for antibody optimization. Thus, there has been significant interest to try to replicate SHM in vitro to create a simple, robust process that would be capable of mimicking the natural processes of affinity maturation directly within a mammalian cellular context to select and evolve antibodies that are immunogenically tolerated, and highly expressed in mammalian cells (Cumbers et al., Nat. Biotechnol., 20(11): 1129-1134 (2002); Wang et al., Prot. Eng. Des. Sel., 17(9): 569-664 (2004); Wang et al., Proc. Natl. Acad. Sci. USA., 101(48): 16745-16749 (2004); Ruckerl et al., Mol. Immunol., 43 (10): 1645-1652 (2006); Todo et al., J. Biosci. Bioeng., 102(5): 478-81 (2006); Arakawa et al., Nucleic Acids Res., 36(1): e1 (2008)).
However, native antibodies that have been isolated from an individual human or animal often fail to demonstrate optimal affinity properties because an intrinsic affinity ceiling inherent in the immune system prevents the in vivo discrimination—and thus selection—of antibodies with affinities more potent than about 100 pM (Batista and Neuberger, Immunity, 8(6): 751-91998, (1998) and EMBO J., 19(4): 513-20 (2000).
The use of phage display libraries can address some of these issues, and phage display based approaches have been shown to be capable of routinely producing high affinity antibodies. However, from a theoretical perspective, such static libraries are inherently limited in their size and scope, because even the largest (1012) libraries can explore only a small fraction of the potential innate immune repertoire. Furthermore it is not possible to simultaneously co-evolve antibodies via phage display approaches on the basis of both good mammalian expression and high affinity, leading to potential downstream manufacturing issues that result from otherwise poor expression in mammalian host cells. Additionally, the use of random mutagenesis in combination with phage display lacks the inherent selectivity profiling found in natural processes of antibody affinity maturation, often resulting in issues of human anti-human immunity, or undesirable cross reactivity profiles.
The use of a cultured cell line to evolve an antibody to a specific target antigen using somatic hypermutation in vitro was first demonstrated using the human Burkitt lymphoma cell line, Ramos (Cumbers et al., Nat. Biotechnol., 20(11): 1129-1134 (2002)). Ramos, and other B cell lines, have also been used successfully to evolve non antibody genes that have been randomly integrated in to the host cell's chromosomal DNA (Wang et al., Prot. Eng. Des. Sel., 17(9): 569-664 (2004) and Proc. Natl. Acad. Sci. USA., 101(48): 16745-16749 (2004)). Additionally, efficient somatic hypermutation has been demonstrated on non antibody genes in B cell lines using episomal vectors, either with or without Ig specific cis regulatory elements (Ruckerl et al., Mol. Immunol., 43 (10): 1645-1652 (2006)). Although some Ramos cell lines show relatively high rates of constitutive hypermutation, B cell lines in general display relatively slow rates of cell division and are difficult to transfect with high efficiency, which limits their practical utility for directed evolution.
The chicken bursal cell line, DT40, diversifies its rearranged Ig light gene by pseudo V gene template gene conversion. However, if gene conversion is blocked by the deletion of the Rad51 paralog, XRCC2 (Sale et al., Nature, 412: 921-6 (2001)), or the deletion of the pseudogene conversion donors (Arakawa et al., Nucleic Acids Res., 36(1): e1 (2008)), the cell line displays constitutive hypermutation in culture. By comparison to Ramos cells, DT40 cells have a significantly shorter generation time (12 hours), are amenable to directed gene targeting and have been successfully used for directed evolution of both endogenous antibodies (Seo et al., Nat. Biotechnol., 23(6): 731-5 (2005); Nat. Protoc., 1(3): 1502-6 (2006); Biotechnol. Genet. Eng. Rev., 24: 179-93 (2007); Todo et al., J. Biosci. Bioeng., 102(5): 478-81 (2006)), and non antibody proteins (Arakawa et al., Nucleic Acids Res., 36(1): e1 (2008)).
While B-cell derivatives such as Ramos and DT40 have been successfully used for directed evolution, the reliable use of these cells in a robust process for directed evolution is complicated by a number of factors including: (i) the need to insert the gene of interest into a defined site in the host cell's Ig locus in order to achieve high level mutagenesis (Parsa et al., Mol. Immunol., 44(4): 567-75 (2007), and (ii) the complex natural biology of somatic hypermutation acting at the endogenous immunoglobulin loci in these cells. Additionally such engineered cell lines exhibit significant clonal instability in SHM rates (Zhang et al., Int. Immunol., 13: 1175-1184 (2001), Martin et al., Proc. Natl. Acad. Sci. USA., 99(19): 12304-12308 (2002) and Nature, 415(6873): 802-806 (2002); Ruckerl et al., Mol. Immunol., 41: 1135-1143 (2004)), and do not provide for any simple means to regulate or control hypermutation, i.e. to switch off mutagenesis after selection of a desired phenotype has been achieved.
The use of non B cells to initiate targeted somatic hypermutation in a gene of interest has been successfully described by a number of groups (Martin et al., Proc. Natl. Acad. Sci. USA., 99(19): 12304-12308 (2002) and Nature, 415(6873): 802-806 (2002); McBride et al., Proc. Natl. Acad. Sci. USA, 103(23): 8798-803 (2006); Jovanic et al., PLoS ONE, 23; 3(1): e1480 (2008); U.S. patent application Ser. No. 09/075,378; International Patent Application Publications WO 08/103474A1 and WO 08/103475A1), and these cell lines can also provide for efficient gene transfer, high level protein expression, optimal growth characteristics and are readily amenable to suspension culture and flow cytometry.
Activation-induced cytidine deaminase (AID) belongs to the APOBEC family of cytidine deaminase enzymes. AID is expressed within activated B cells and is required to initiate somatic hypermutation (Muramatsu et al., Cell, 102(5): 553-63 (2000); Revy et al., Cell, 102(5): 565-75 (2000); Yoshikawa et al., Science, 296(5575): 2033-6 (2002)) by creating point mutations in the underlying DNA encoding antibody genes (Martin et al., Proc. Natl. Acad. Sci. USA., 99(19): 12304-12308 (2002) and Nature, 415(6873): 802-806 (2002); Petersen-Mart et al., Nature, 418(6893): 99-103 (2002)). AID is also an essential protein factor for class switch recombination and gene conversion (Muramatsu et al., Cell, 102(5): 553-63 (2000); Revy et al., Cell, 102(5): 565-75 (2000)).
The discovery that AID is responsible for initiating somatic hypermutation has opened the possibility of using non B cell lines to create more defined, stable and controllable systems for utilizing somatic hypermutation.
Despite these advances, key challenges regarding the development of a practical system for somatic hypermutation remain, including (1) the ability to target somatic hypermutation to a gene of interest, and away from structural genes, (2) the relatively low rates and nature of the mutations achieved using exogenous AID compared to somatic hypermutation in vivo, and (3) the relatively long cell doubling times required to grow up a cell population from a single cell clone between cycles of mutagenesis.
Thus, there is a specific need for improved compositions and methods to improve the efficiency of somatic hypermutation systems. This invention provides such compositions and methods.